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Pauline in the Game Boy version of Donkey Kong, which marked the debut of her revised appearance. This, and her appearance in Mario vs. Donkey Kong were used for her trophies in Smash 4.

Pauline was Mario's original damsel-in-distress and love interest, appearing under the name "Lady" in the Donkey Kong on arcade machines and its subsequent versions. In her original appearance, she is a blonde with a pink dress. Lady's hat, purse, and parasol (as well as sometimes a birthday cake), which she ostensibly dropped after being captured by Donkey Kong, appear as collectibles for extra points. As Mario pursues him, Donkey Kong drags Lady farther and farther up the building until he is finally defeated and she is set free and reunited with Mario. Lady appears in all versions of the game; however, she is replaced with Princess Peach, who has similar blonde hair and a pink dress, in Super Mario Bros. and subsequent Mario games.

In the Donkey Kong remake for Game Boy, Pauline returns under her new name and a new appearance, differentiating her from her successor with brown hair and a red dress. Her role in the game is largely the same, though Donkey Kong is now joined by his son, Donkey Kong Jr..

After over a decade of making cameos or appearing in minor ports of the original Donkey Kong, Pauline made her return to the series in Mario vs. Donkey Kong 2: March of the Minis, where she joins Mario at the grand opening of his new theme park. As usual, Donkey Kong becomes infatuated with her, but she rejects him in favor of Mario, sending him into a rage and leading him to kidnap her; however, this time, Mario utilizes his toy Mini Marios to rescue her. After she is saved, she and Mario make peace with Donkey Kong. In the following game, Pauline is kidnapped once more, but this time when Mario and the Mini Marios save her, it turns out that her kidnapping was a ruse she orchestrated to test the toys' skills. In Mario vs. Donkey Kong: Mini-Land Mayhem!, a Mini Pauline is added to the roster of toys, which Donkey Kong immediately becomes enthralled by, becoming furious when they sell out before he can get one and kidnapping Pauline once more, though he lets her go once Mario gifts him with his own Mini Pauline. Pauline is not kidnapped in Mario vs. Donkey Kong: Minis on the Move, instead running different arcade games alongside Donkey Kong peacefully. In Mario vs. Donkey Kong: Tipping Stars, her apparent kidnapping again turns out to be a ruse, this time to lure Mario to a surprise party. The Mini Pauline also returns in the latter two games.

Finally, Pauline makes her mainstream Mario debut in Super Mario Odyssey, where she is the mayor of the Metro Kingdom, the capital of which, New Donk City, is based on the city setting of the original Donkey Kong. When Mario first arrives in New Donk City, it is under attack by Bowser's Mechawiggler, with Pauline watching the destruction from afar, unable to do anything about it. After Mario defeats the Mechawiggler, Pauline is able to swiftly repair the city and plans a festival to celebrate things returning to normal. She asks Mario to help her gather a band so that she may perform a song at the festival, and Mario complies. However, before the festival can occur, Mario must investigate the power plant malfunctioning, and Pauline joins him upon realizing the sabotage is Bowser's fault. As thanks for helping make the festival possible, Pauline declares Mario the guest of honor. The song she and her band perform, Jump Up, Super Star!, also serves as the game's main theme song. Later, Pauline is among the guests kidnapped by Bowser to attend his and Peach's wedding, telling Mario he is just in time when he arrives. Pauline can be heard singing a second song, Break Free (Lead the Way), as Mario and Peach escape the collapsing wedding hall at the end of the game. Once Bowser is defeated, Mario can visit Pauline in New Donk City, where she will reveal several facts about herself, including that her hat is her favorite possession, she loves eating cake, and that being kidnapped by Donkey Kong was "traumatic". Mario can also give Pauline a purse as a birthday gift, revealing that she lost her purse and never replaced it when Donkey Kong kidnapped her. Pauline also appears among other characters on the Darker Side of the Moon, performing Jump Up, Super Star! and cheering Mario on as he enters the final challenge.

Following her appearance in Super Mario Odyssey, Pauline was also added as a playable character to Mario Tennis Aces in a free update.

Waaay back in 1981, a new action game using barrels and girders hit arcades. That game was Donkey Kong, in which Mario had to rescue Pauline from the title character. Maybe she wanted to look less like Princess Peach—she changed her look in the Game Boy version.

One fine day in 1981, the world was introduced to a new action-packed arcade game - Donkey Kong! But back then, the girl Mario was trying to rescue from Donkey Kong wasn't Peach - it was Pauline. Also, did you know that in Japan, Pauline was originally just called "Lady"?

Two different spirits also exist in her image, one featuring her artwork from Super Mario Odyssey and the other featuring her artwork in the clutches of Donkey Kong from the original Donkey Kong and referring to her by her original name, "Lady".